【作者】 张亮；

【导师】 杨学军；

【作者基本信息】 天津医科大学 ， 外科学 神外（专业学位）， 2021， 博士

【摘要】 目的高级别脑胶质瘤具有生长更快、更具侵袭性的特点,其中GBM的发病率最高、预后最差,尽管手术联合放疗和化疗的综合治疗模式已让部分高级别胶质瘤患者获益,但作为一线化疗药物TMZ存在耐药性问题影响了治疗效果,耐药机制在不断的被研究、发掘,本研究拟首先阐明TMZ在GBM中发挥抗肿瘤过程中新的作用机制及进一步研究是否存在新型miRNA效应靶标;再针对目前在肿瘤研究中的Wnt/β-catenin信号途径及KLF4因子是否存在并参与胶质瘤进展过程,结合生物信息学分析,以及生理功能验证,讨论miR-325,miR-596以及miR-32在GBM中的作用和调控机制;以期为后续进一步开展研究miRNA在提高GBM对TMZ化疗敏感性及筛选效应靶标提供实验基础。1.探究替莫唑胺是否存在通过miRNA途径发挥抗脑胶质瘤的作用及其新型效应靶标;2.研究miR-325及靶标基因TFRC在抑制胶质瘤细胞增殖和侵袭中的作用和潜在机制;3.研究miR-596及其靶标基因CREPT和在抑制胶质瘤细胞恶性生物学行为中的作用。4.研究miR-32及其靶标基因KLF4在抑制胶质瘤细胞恶性生物学行为中的作用。实验方法1.通过质粒转染方法过表达或敲低相关目的基因到相应胶质瘤细胞。2.通过RT-qPCR对相关基因在不同条件下mRNA表达水平变化进行评估。3.通过Western blot对相关基因在不同条件下蛋白表达水平变化进行评估。4.通过MTT测定评估胶质瘤细胞活力。5.通过CCK-8以及克隆形成测定对胶质瘤细胞增殖能力进行评估。6.通过Transwell对不同条件下胶质瘤细胞的侵袭能力进行分析。7.通过细胞划痕实验评估胶质瘤细胞的迁移能力;8.通过生物信息学手段对miRNA的潜在靶标基因进行分析预测。9.通过双荧光素酶报告基因检测确认miRNA的靶标基因。10.使用SPSS19.0软件对实验数据进行统计学分析。结果1.替莫唑胺通过抑制TFRC表达抑制胶质瘤细胞增殖和侵袭:替莫唑胺在一定剂量范围内以剂量依赖的方式通过抑制TFRC在胶质瘤U87细胞中的表达抑制胶质瘤U87细胞增殖和侵袭;TFRC的过表达减少替莫唑胺对胶质瘤U87细胞增殖和侵袭的抑制作用;TFRC的低表达增加替莫唑胺对胶质瘤U87细胞增殖和侵袭的抑制作用。2.miR-325通过靶向调控TFRC抑制胶质瘤细胞增殖和侵袭:TFRC是miR-325的靶标基因并受其负调控;miR-325在胶质瘤组织和细胞系中被下调并与患者存活率相关;通过上调miR-325抑制了TFRC表达以抑制胶质瘤U87细胞和U251细胞的增殖和侵袭;通过下调miR-325表达使得TFRC过表达促进了胶质瘤U87细胞和U251细胞的增殖和侵袭。3.CREPT通过激活Wnt/β-catenin途径促进胶质瘤细胞增殖和侵袭并作为miR-596的靶标基因:CREPT的沉默抑制胶质瘤LN-18细胞和U-138MG细胞增殖和侵袭;CREPT是miR-596的靶标基因并受其负调控;miR-596调控胶质瘤LN-18细胞和U-138 MG细胞增殖和侵袭;miR-596的过表达或CREPT的沉默抑制Wnt/β-catenin通路的激活;CREPT的过表达降低了miR-596对胶质瘤LN-18细胞和U-138 MG细胞的增殖和侵袭的抑制作用。4.miR-32通过靶向抑制KLF4的表达促进胶质瘤增殖和侵袭:miR-32促进胶质瘤U87细胞增殖、侵袭和迁移;KLF4是miR-32的靶标基因并受其负调控;KLF4抑制胶质瘤U87细胞增殖、侵袭和迁移;KLF4的低表达缓减了miR-32的下调对胶质瘤U87细胞的增殖和侵袭的抑制作用。结论1.TFRC是替莫唑胺在GBM中发挥抗肿瘤作用的新型效应因子;miR-325通过下调TFRC表达抑制胶质瘤进展。表明miR-325新型靶基因TFRC是一种癌基因。2.miR-596通过靶向CREPT抑制其介导的Wnt/β-catenin通路来抑制胶质瘤细胞的增殖和侵袭,miR-596/CREPT/Wnt/β-catenin信号轴在胶质瘤发生过程中发挥着重要作用。miR-596通过靶向抑制CREPT的表达抑制胶质瘤细胞增殖和侵袭。miR-32通过靶向抑制KLF4的表达促进胶质瘤细胞增殖和侵袭。miR-596新型靶基因CREPT是一种癌基因。miR-32新型靶基因KLF4是一种抑癌基因。更多还原

【Abstract】 Objective:TMZ is a first-line chemotherapeutic agent for high-grade glioma,but the problem of drug resistance is becoming more and more prominent,this study aims to first clarify whether there are new effective targets in the anti-tumor action of TMZ in GBM.Then,the Wnt/β-catenin pathway and KLF4,which are still to be further studied in GBM,were combined with bioinformatics analysis and physiological function verification to discuss the interaction and regulation mechanism of miR-325,miR-596 and miR-32 in GBM.It is expected to provide an experimental basis for further studies on the role of miRNAs in enhancing the sensitivity of GBM to TMZ chemotherapy.1.Aim to explore whether temozolomide has an anti-glioma process through the miRNA pathway,and its new effect targets;2.To study the role and potential mechanism of miR-325 and the target gene TFRC in inhibiting the proliferation and invasion of glioma cells;3.To study the role of miR-596 and its target gene CREPT in inhibiting the malignant biological behavior of glioma cells.4.To study the role of miR-32 and its KLF4 target gene in inhibiting the malignant biological behavior of glioma cells.Methods:1.Cell transfection was performed to overexpress or silence related genes;2.RT-qPCR was performed to detect the levels of related genes;3.Western blot was performed to detect the levels of related proteins;4.MTT assay was performed to detect the viability of glioma cells;5.CCK-8 or colony formation assay were conducted glioma cell proliferation;6.Transwell was conducted to assess glioma cell invasion;7.Wound healing migration assay was performed to assess glioma cell migration;8.Bioinformatics was performed to predict the potential target genes of microRNAs;9.Dual luciferase reporter gene assay was performed to confirm the target genes of microRNAs;10.SPSS19.0 software was performed for statistical analysis of experimental data.Results:1.Temozolomide inhibits proliferation and invasion of glioma cells by inhibiting TFRC expression: Temozolomide inhibits proliferation and invasion of glioma U87 cells by inhibiting TFRC expression in a dose-dependent manner in a certain dose range,and inhibits proliferation and invasion of glioma U87 cells;Overexpression of TFRC alleviates the inhibitory effect of temozolomide on proliferation and invasion of U87 cells in glioma.TFRC silences aggravate the inhibitory effect of temozolomide on the proliferation and invasion of glioma U87 cells.2.miR-325 inhibits glioma cell proliferation and invasion by targeting TFRC: TFRC is a target gene of miR-325 and is negatively regulated by TFRC.miR-325 was down-regulated in glioma tissues and cell lines and correlated with patient survival rate.By up-regulating miR-325,TFRC expression was inhibited to inhibit proliferation and invasion of glioma U87 and U251 cells.Over-expression of TFRC promoted proliferation and invasion of glioma U87 cells and U251 cells by downregulating miR-325 expression.3.CREPT through the activation of Wnt/β-catenin pathway to promote glioma cell proliferation and invasion and as target genes of miR-596: CREPT silence suppression glioma LN-18 cells and U-138 MG of cell proliferation and invasion;CREPT target genes of miR-596 and is subject to the negative control;miR-596 regulates proliferation and invasion of glioma LN-18 cells and U-138 MG cells.CREPT in silence or miR-596 expression inhibition of Wnt/β-catenin pathway activation;The overexpression of CREPT reduced the inhibitory effect of miR-596 on the proliferation and invasion of glioma LN-18 cells and U-138 MG cells.4.miR-32 promoted the proliferation and invasion of glioma by targeting KLF4expression: miR-32 promoted the proliferation,invasion and migration of glioma U87cells;KLF4 is a target gene of miR-32 and is negatively regulated by it.KLF4 inhibited the proliferation,invasion and migration of glioma U87 cells.Silencing of KLF4 attenuates the inhibition of miR-32 down-regulation on the proliferation and invasion of glioma U87 cells.Conclusion:1.TFRC is a novel effector of temozolomide in GBM,and also a target gene of miR-325;miR-325 inhibits glioma progression by down-regulating TFRC expression.This suggests that miR-325 targets TFRC as a tumor suppressor gene.2.miR-596 inhibits the proliferation and invasion of glioma cells by targeting CREPT by inhibiting the Wnt/β-catenin pathway mediated by miR-596,and the miR-596/Wnt/β-catenin signaling axis plays an important role in the process of glioma.miR-596 inhibited proliferation and invasion of glioma cells by targeting the expression of CREPT.miR-32 promotes proliferation and invasion of glioma cells by targeting KLF4 expression.The novel target gene of miR-596 CREPT is an oncogene.The novel target gene of miR-32 KLF4 is an contra-oncogene.更多还原